Ballistic transformation of C. elegans

ABSTRACT

A ballistic method of introducing nucleic acid into a nematode worm is described which involves bombarding the nematode with a plurality of microprojectiles. Nematode worms transformed according to the method of the invention are also provided.

[0001] The invention is concerned with a method of introducing nucleicacid into nematode worms, in particular Caenorhabditis elegans.

[0002] Transgenic Caenorhabditis elegans (C. elegans) are currently madeby injecting nucleic acid (usually DNA) into the hermaphrodite gonad(i.e. into a syncitium)or into individual oocyte nuclei. Typically oneinjects a mixture of the DNA one wants to introduce (hereinafterreferred to as ‘test DNA’) and a plasmid carrying a selectable markerthat allows one to distinguish transgenic progeny from non-transgenicprogeny. The selectable marker can be a visible phenotypic marker whichleads to a change in shape or movement of the transgenic worms (e.g.rol-6), a marker rescuing a conditionally lethal gene introduced intothe genetic background of the injected worms or a plasmid containingnucleic acid encoding green fluorescent protein (GFP) from the jellyfishAequorea Victoria. The offspring of injected worms (F1 generation) arethen screened for animals expressing the selectable marker.

[0003] The F1 offspring of an injected hermaphrodite typically containon average 1 to 10 individuals that express the selectable marker. Theseindividuals are then placed in culture but on average only 10% will goon to transmit the selectable marker to their offspring. One generallyassumes that when the marker DNA is accepted into the worm genome andtransmitted to the offspring, the test DNA which one wants to introduceis co-transformed.

[0004] This current method of transformation has practical limitationsin that introducing DNA into worms entails one by one manipulation andinjection of syncitia/oocytes under a microscope. This work is timeconsuming and requires a considerable amount of expertise. It istypically possible for a person skilled in the injection technique toinject ˜50 hermaphrodites in a single day.

[0005] Transgenic worms transmitting the transformed DNA in a heritablemanner have incorporated an extra minichromosome, consisting of amixture of marker and test DNA linked together in an unpredictablestructure. This minichromosome is mitotically and meiotically unstableand is lost at a rate of 1% to 99% per cell division.

[0006] It is an object of the present invention to provide a moreefficient transformation system for C. elegans. Ideally one would wishto achieve integration of the exogenous test DNA by homologousrecombination with the C. elegans chromosome. In order to achieve thisaim it will be necessary to develop a technique by which DNA can besimultaneously introduced into a large number i.e. thousands ofindividual worms.

[0007] A recently developed method for introducing DNA into cellsinvolves shooting cells with microprojectiles, typically gold ortungsten particles of around 2 μm diameter, which have been coated withthe DNA to be introduced. This technique, generally known to personsskilled in the art as ballistic transformation, has been used tosuccessfully deliver DNA into plant cells (Klein et al. Nature, 327:70-73 (1987); Christou et al. Plant Physiol., 87: 671-674 (1988);Takeuchi et al. Plant Molecular Biology, 18: 835-839 (1992)), culturedmammalian cells (Zelenin et al. FEBS Letters, 244: 65-67 (1989)),fertilized fish eggs (Zelenin et al. FEBS Letters, 287: 118-120 (1991))and intact mouse tissues and organs (Zelenin et al. FEBS Letters, 280:94-94 (1991); Williams et al. Proc. Natl. Acad. Sci. USA, 88: 2726-2730(1991)).

[0008] Despite the success of the technique with plant cells andcultured mammalian cells problems have been anticipated by those skilledin the art in applying ballistic techniques to nematode worms. However,the present inventors have successfully applied a similar ballistictransformation technique to the introduction of nucleic acid into C.elegans. Using this technique it is possible to introduce nucleic acidsimultaneously into a large number of individual worms.

[0009] Accordingly, in a first aspect the invention provides a method ofintroducing nucleic acid (DNA and/or RNA) into a nematode wormcomprising bombarding the worm with a plurality of microprojectiles.

[0010] In one embodiment of the invention the microprojectiles arecoated with the nucleic acid which it is desired to introduce into thenematode worm.

[0011] Bombardment of the nematode worm with high velocitymicroprojectiles is achieved using a particle bombardment gun based onflowing helium of a type known in the art, see for example Johnston,Nature, 346: pp776; Klein et al. Biotechnology, 10: pp286-291 andTakeuchi et al. Plant Mol. Biol., 18: pp835-839. The gun uses a flowingstream of helium gas to accelerate DNA coated particles towards a targetsample to be transformed.

[0012] A detailed protocol for ballistic transformation of C. elegansusing nucleic acid coated microprojectiles is described in the examplesgiven herein. Briefly, a small pellet of worms is dispensed onto a smallnematode agar plate. The plate is then placed inside the ‘gun’ and asuspension of microprojectiles (e.g. gold particles) coated with nucleicacid is shot at the worms. After a short recovery period the plate iscut into a number of segments which are placed on large agar plates togrow worms for selection of transgenic animals. The transformationprocedure takes only a few minutes and is technically very simple sothat a large number of experiments can be undertaken in very littletime.

[0013] In an alternative embodiment of the method of the inventionballistic transformation can also be accomplished by first applying asolution containing the nucleic acid directly onto the nematodes andthen shooting the nematodes with ‘bare’ microprojectiles which have notbeen coated with nucleic acid. With this technique it is not necessaryto coat the microprojectiles with nucleic acid. Using the conventionalbombardment technique (i.e. using coated microprojectiles) transformedoffspring are produced as a result of a coated particle being fired intoa gonad cell of the worm. Using the alternative approach, in which theworms are first coated with a dense solution of nucleic acid and thenbombarded with ‘bare’ microprojectiles, a particle may drag the DNAalong its passage through the worm and hence the particle does notnecessarily need to stop within a gonad cell. If the particle merelypasses through a gonad cell on its passage through the worm it may leavebehind a sufficient amount of the nucleic acid it is dragging along toresult in transformation of the gonad cell.

[0014] In order to facilitate selection of transformants into which DNAhas been successfully introduced by the method of the invention it ispreferred to use a dual selection protocol using a dominant phenotypicmarker such as, for example, rol-6 or an autonomous fluorescent protein(AFP) in combination with a marker rescuing a conditionally lethal geneintroduced into the genetic background of the injected worms. As usedherein the term “autonomous fluorescent protein” encompasses both greenfluorescent protein (GFP) and blue fluorescent protein (BFP) and anyother autonomous fluorescent protein of this type. The examples givenbelow relate to the transformation of C. elegans with a geneticbackground carrying a temperature sensitive mutation in the pha-1 genewherein DNA encoding the wild-type pha-1 gene is introduced as aco-selectable marker. However, other conditional lethal mutations couldhave been used with equivalent effect and it is to be understood thatthe present invention is not to be limited by the nature of theselectable markers employed to facilitate the identification oftransformed worms.

[0015] The present invention will be further understood with referenceto the following Examples, together with the accompanying Figure.

[0016]FIG. 1 shows a Normarski micrograph of C. elegans which have beenbombarded with gold particles. The upper arrow points at a gold particlelocated in the gonad, the lower arrow on one located in the intestine ofa young hermaphrodite.

EXAMPLE 1 Basic Protocol for Ballistic Transformation

[0017] (A) Synchronised worm culture

[0018] 1. C. elegans worms (strain pha-1(e2123ts)) were grown on largestandard NGM-plates to starvation to promote accumulation of larvae ofthe L1 stage.

[0019] 2. Pieces of agar containing ‘L1-islands’ were cut out and usedto inoculate fresh large NGM-plates.

[0020] 3. The worms were grown up to the young adult stage at 15-20° C.depending on the requirements of the particular C. elegans strain inclean pseudo-sterile surroundings.

[0021] 4. Worms were washed off the plates with distilled water oregg-buffer, pooled in 50 ml Falcon tubes and allowed to sediment bygravity.

[0022] 5. Approximately 500-800 μl aliquots of the worm pellet wereaspirated using a Gilson® pipette equipped with a blue tip and placeddropwise in the centre of small NGM-plates.

[0023] 6. The plates were placed on ice and the liquid allowed to soakin leaving back a heterogenous worm “pillow”. The worm pillow was formedinto a circular shape of approximately 10 mm diameter using a platinumspatula and left on ice until use.

[0024] (B) Ballistic particle bombardment

[0025] 1. An NGM plate containing the ice-cooled worms was placed on thecrosshair table within the vacuum chamber of the gun, with a distance of120 mm to the opening of the shooting chamber. The lid of the NGM platewas removed and the door of the vacuum chamber immediately closed. Thesteel grid within the shooting chamber was then loaded with DNA-coatedgold suspension (in ethanol). The gold particles were coated with amixture of test DNA and marker DNA (plasmids pRF4 containing rol-6 andpBX containing wild-type pha-1).

[0026] 2. The helium pressure presetting valve was set to 8-10 bar. Thevacuum chamber was then evacuated, the pressure discharge being releasedwhen the partial vacuum reached a pressure of −50 to −100 mbar.

[0027] 3. The door of the vacuum chamber was then opened and the lid ofthe NGM plate immediately replaced to preserve sterility. The plate wasthen placed at 15° C. to allow the worms to recover from the bombardmentprocedure.

[0028] 4. The NGM agar was cut into 4-8 segments and each segment placedon a fresh large enriched NGM plate (double tryptone). The large plateswere then incubated at 15-20° C.

[0029] (C) Selection for transformants

[0030] After 6 to 7 days post-transformation the F1 worms were screened:

[0031] (i) visually for worms expressing the rol-6 phenotype, thoseexpressing the rol-6 phenotype being isolated and subsequently testedfor stable transformation, and/or

[0032] (ii) by shifting to 25° C. and maintaining in culture for afurther 3 to 4 days to screen for pha-1 rescue, and/or following eitherof the above

[0033] (iii) individual transgenic worm lines both expressing the rol-6phenotype and exhibiting pha-1 rescue at 25° C. were then individuallytested for the presence of the test DNA using techniques known in theart.

EXAMPLE 2 Detailed Protocol for Ballistic Transformation

[0034] The methods used are described in the form of a recipe. All stepsare carried out under sterile conditions. General C. elegans methods aredescribed in Wood W. B. (1988) ‘The nematode Caenorhabditis elegans’,Cold Spring Harbor Laboratory, New York.

[0035] (A) Preparation of worms.

[0036] Grow target worm strain (here pha-1 (e2123ts)) on large standardNGM-plates (90 mm diameter) to starvation so that plates are coveredwith many islands of L1 larvae. Depending on the size of the‘L1-islands’ cut out agar pieces of 5-10 mm² and inoculate about 8 freshlarge NGM plates per 10 shots. Worms should not starve before they reachadulthood. The worms can be fed on bacteria such as E. coli. Plates areready when about 50% of the worms contain a few eggs. Wash the worms offthe plates with distilled water and pool in 50ml tubes. Let the wormssediment down by gravity (˜15 min at room temperature). Approximately100 μl of the worm pellet are placed in the centre of small (35 mm)NGM-plates. These plates have been dried for several days and are seededthe day before (incubation at RT) with a thin layer of E. coli (strainOP50) with diameter of approximately 10 mm. Put the plates back on iceto stop the worms from moving about and let the liquid soak up leavingback a heterogeneous worm “pillow”. Take a platinum spatula and form amore or less homogeneous and circular shaped worm “pillow” with adiameter of about 10 mm. Leave on ice until use; not longer than 1-2hrs.

[0037] (B) Ballistic Particle bombardment.

[0038] The bombardment device (gun) is calibrated by shots at a filterpaper placed at shooting distance (see below) and subsequent drawing ofa crosshair through the centre of the target area. Calibration should berepeated from time to time. Set the He pressure pre-setting valve to8-10 bar for calibration and transformation. Place and adjust theice-cooled worm plate on the crosshair table within the gun vacuumchamber with a distance of 120 mm to the filter holder, take off theplate's lid only before closing the chamber. Load the steel grid withinthe shooting chamber with the DNA-coated gold suspension in EtOH. Startto evacuate the vacuum chamber and trigger the gun (pulse timeapproximately 10 ms; the pressure wave should not release the lid of thedevice for pressure release) when the partial vacuum reaches values of50 to 100 mbar.

[0039] We were not able to determine settings which gave significantlythe best result for either the bombardment pressure or the partialvacuum. A systematic analysis of this might be useful for an individualapparatus. Worms survived even stronger partial vacuum and might also betransformable with less then 8 bar pressure.

[0040] Release the vacuum and immediately close the worm plate again.Allow the worms to recover at 15° C. for approximately 30 minutes. Wormswill warm up and start moving again. Cut the agar into 4 or more sectorsand put each piece on a fresh 90mm enriched NGM-plate (double tryptone).Leave plates at 15-20° C. depending on the selection protocol fortrangenes.

[0041] (C) Screening procedure.

[0042] This procedure may vary depending on the actual system used toscreen for transgenic worms. In case of using rol-6 (Mello, C. C. et al.(1991) EMBO J. 10: 3959-3970) and /or pha-1 (Granato, M. et al. (1994)Nucl. Acids Res. 22: 1762-1763):

[0043] Search for rol-6 animals among the F1 generation after 6-7 days(15° C.). Rol animals are placed on individual plates and subsequentlytested for stable transformation. Stable transgenic lines should producerol-6 offspring in a non-Mendelian ratio. The remaining F1 generation isshifted to 25° C. and tested for pha-1 rescue after another 3-4 days.Rescue is indicated by the appearance of young F2 larvae on the plates.Check plates again after another 3-4 days. If viable worms are found,10-20 of these F2 or F3 animals are individually tested for stabletransformation by non-Mendelian segregation of dead eggs and viableworms. It should be noted that pha-1 reverts occasionally by acquiringspontaneous second site suppressors (Schnabel, H. et al. (1991)Genetics, 129: 69-77). Therefore the pha-1 strain should be checkedregularly for its integrity by shifting some worms to 25° C., wherepha-1 produces only dead eggs. The presence of the ceh-13 GFP reporterconstruct was tested directly by viewing embryos from transgenichermaphrodites under a fluorescence microscope. To test for rescue ofthe non-conditional allele t1237 of the maternal effect embryonic lethalgene sud-1 the transgenic array was crossed into a sud-1 background(vab-9 sud-1 (t1237)/mnc1; lon-2) and the progeny of homozygous Vabhermaphrodites (vab-9 sud-1) was screened for viability.

[0044] (D) Preparation of gold particles for ballistic bombardment.

[0045] The recipe is based on the method of Takeuchi et al. (1992) PlantMol. Biol., 18: 835-839. 5mg gold powder (Au; powder, spherical, Ø 1.5-3μm; Aldrich®) is placed in an 1.5 ml Eppendorf tube and the particlesare prewashed with 500 μl distilled water; a homogeneous suspensionshould appear after vortexing. Let the gold particles sediment anddiscard the water carefully. Add a small volume of fresh distilled waterand 20 μg of each plasmid DNA. Adjust volume to 180 μl with water andadd 20 μl of a 3M Na-acetate solution. Vortex. The DNA is precipitatedwith 2.5 volumes EtOH. Store for 30 min at −20° C. Vortex several timesduring this period. Settle the Gold particles by gravity and aspiratethe supernatant. Do not centrifuge. Suspend the particles in 200 μlice-cold absolute EtOH. Vortex. Particles can now be stored at −20° C.For transformation load 20 μl (approximately 2-6 μg of DNA per shot) ofthe suspended solution in the filter paper of the Swinny®-Filterholder(Millipore) used for bombardment. Mount and shoot immediately.

[0046] An alternative method for preparing the Gold particles is asfollows:

[0047] Add 20 μg of plasmid DNA to 10 mg Gold powder (Au; powder,spherical, Ø 1.5-3 μm; Aldrich®). Add distilled water to a total volumeof 200 μl then add 20 μl 3M Na-acetate and 550 μl ethanol and place at−20° C. for at least three hours, with vigorous vortexing every 30minutes, to precipitate the DNA. After 3 hours let the Gold particlessediment, aspirate the supernatant and re-dissolve the Gold particles in200 μl ice-cold ethanol. 20 μl of this solution is used for eachexperiment.

[0048] (E) Preparation of glass particles for ballistic bombardment.

[0049] As an alternative to Gold particles ballistic transformation mayalso be performed with glass microprojectiles prepared as follows:

[0050] Add 20 μg plasmid DNA to 10 μl of ‘glassmilk’ activated glasssuspension (Jetsorb) and add 300 μl buffer Al (supplied with theglassmilk suspension by the manufacturer). Allow the DNA to bind to theglassmilk for 15 minutes at 53° and after centrifugation wash the pelletwith 300 μl buffer A1. The resultant suspension is re-pelleted bycentrifugation then washed twice with buffer A2 (also supplied by themanufacturer with the glassmilk suspension). After washing the pellet isdried and resuspended in 200 μl of ethanol. 20 μl aliquots are used foreach transformation experiment, following the ballistic bombardmentprocedure as described above for Gold particles. Other activated glasssuspensions from other manufacturers can also be used in this procedure.

[0051] Although activated glass suspensions are known to bind DNA,probably even better than gold, ballistic transformation experimentswith glass particles did not result in enhanced transformationefficiencies. Although probably more DNA is bound to the beads, glasshas a lower density than gold and will probably be less efficient topenetrate the nematode. Nevertheless, it is possible to transform C.elegans with glass particles at approximately the same efficiencies aswith gold particles, indicating that neither the quantity of introducedDNA nor the density of the particle is of major importance.

RESULTS

[0052] The results of a number of independent ballistic transformationexperiments using Gold particles, performed according to the protocolsdescribed above, are summarised below. In each case the nature of thetest DNA and marker DNA is given. For each test DNA/marker combination anumber of different bombardment procedures were performed according tothe method given in part (B) above. Transformants were then scored forexpression of rol-6 phenotype and rescue of the pha-1 conditional lethalmutation.

[0053] In general, using rol-6 selection an average of two transformantsper shot was obtained by scoring the F1 for rolling animals on the largeplates derived from each shot (step 4 of part (B)). As with themicroinjection protocol only 10% of these were stable during the nextgenerations (one line in 6 shots). These lines always also expressed twoother cotransformed plasmids (pha-1 in conjunction with either aGFP-reporter construct of ceh-13 (Wittmann, C. et al. (1997)Development, 124: pp4193-4200) or a plasmid containing the sud-1 gene,see the results summarised in Table 10A. Using the pha-1 selectionsystem approximately one stable line per two shots was obtained byshifting the plates to the non-permissive temperature for rescue in theF2 generation. The pha-1 selection system is thus three times moreeffective than the rol-6 system in selecting transformed animals. Thepha-1 transgenic animals also co-expressed a second marker, however,co-transformation occurred with a slightly lower frequency (70%) than inthe animals from the same shots selected for stable rolling after thefirst generation (Table 10B). It is possible that the co-transformationdepends initially on a critical amount of DNA so that the different DNAsare reliably co-ligated to form concatemeric arrays (Mello, C. C. et al.(1991), EMBO J., 10: pp3959-3970). By selecting with the less sensitiverol-6 phenotype one may just miss animals below a certain threshold andthus select for animals which had a higher chance for co-ligation.

[0054] Table 11 shows a detailed analysis of a series of shots. Acertain clustering of the transformation events was observed. Nine outof the fourteen plates with lines transformed for pha-1 in the F2selection carried Rol animals in the F1 selection. Also, stable linesfor both markers discovered in the Rol selection (F1) came in clusters.These plates always harboured additional doubly transformed lines in theF2 selection. However, this could be due to a failure to find allrolling animals in the F1 selection. It is not clear how manyindependent events are hidden in the F2 selection.

[0055] In the examples given herein an effective distance of 120 mm anda pressure of 8 to 10 bar was used for all shots. Variation of pressure(6 to 10 bar) and distance was observed to have very little effect onthe efficiency of transformation. When plates were viewed under amicroscope after a shot many worms in the centre of the plate were foundto be killed and the worms around this zone contained gold particles(FIG. 1) while worms in the outer zone did not. It thus seems that thevelocity gradient is very steep and under the conditions of the examplesused herein there is a narrow zone of transformation which may come tolie in different positions within the worm pellet depending on pressureand distance. An important factor is to place the worms in a thin andfairly dry bacterial lawn, otherwise the worms are blown away.Concerning the amount of worms used per shot, sufficient worms were usedto span the shooting area but not too many worms as it becomes awkwardto handle too many F1 worms for the Rol selection or an even largernumber of F2 worms for pha-1 selection.

[0056] Variation in the efficiency of the transformation procedure isobserved to occur even in repeat experiments in which no adaptation ismade to the protocol i.e. using identical amounts of DNA, the samesettings on the gun and identical culture conditions for the nematodes.Closer observation shows that bombardment experiments in which the wormsremained in the centre of the plate after bombardment resulted in highertransformation efficiencies than experiments in which the worms wereblown away from the centre of the plate. Moreover, worms that are blownaway by the high pressure of the gun did not survive the bombardmentprocedure.

[0057] The reproducibility and, to a lesser extent, the efficiency ofthe ballistic transformation procedure can be improved by using very dryagar plates, agar plates containing a high concentration of agar andagar plates which have not been seeded with E. coli. Furthermore,immobilization of the worms on the agar plate will also result inenhanced efficiency and reproducibility of the ballistic transformation.

[0058] In summary, it has been demonstrated that C. elegans can betransformed by ballistic bombardment. At present the method is about asefficient as the microinjection procedure which, however, depends muchmore on the training and skill of the person carrying out the procedureand on much more expensive equipment. Unlike microinjection, wheredehydration of the worms helps to relieve the internal pressure of thenematode and thus avoids bursting when they are penetrated by theneedle, no ballistic transformation was achieved with dehydrated worms.

SHOT EXPERIMENT: 1

[0059]C. elegans-strain: pha-1 (e2123)

[0060] Marker-DNA:

[0061] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0062] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0063] Test-DNA: —

[0064] Addition of nucleic acid:—Status: concluded TABLE 1 results ofshot experiment 1 current 1. selection (rol-6) Test-DNA 2. selection(pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1

2 3

X 4 5

X_(Rol) X X_(Rol) X_(Rol) 6

7

X_(Rol) X_(Rol) 8 2_(pha) X_(Rol) 9 X_(Rol) 10

11

12

X_(Rol) 13

14

15 16 7_(pha) X_(Rol) 17

10_(pha) X_(Rol) X_(Rol) X_(Rol) 18

SHOT EXPERIMENT: 4

[0065]C. elegans-strain: pha-1 (e2123)

[0066] Marker-DNA:

[0067] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0068] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0069] Test-DNA: pH1-FM6.9 (sud-1) after crossing with vab-9 sud-1

[0070] Addition of nucleic acid:—Status: concluded TABLE 2 results ofshot experiment 4 2. selection current 1. selection (rol-6) Test-DNA(sud-1) (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2 X 3

4

X 5

6 7

2_(pha) X_(Rol/pha) 8 9

10

1_(pha) X_(Rol/pha) 11 12 13 14 15 16

17

18 19

20

SHOT EXPERIMENT: 6

[0071]C. elegans-strain: pha-1 (e2123)

[0072] Marker-DNA:

[0073] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0074] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0075] Test-DNA: pH1-FM6.9 (sud-1) after crossing with vab-9 sud-1

[0076] Addition of nucleic acid:—Status: concluded TABLE 3 results ofshot experiment 6 current 1. selection (rol-6) Test-DNA (sud-1) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1

2 X 3

4 5

X 6

7

X_(Rol) 8

9 10

11

12 13

14 15

16

4_(pha) X_(Rol/pha) X X_(Rol) 17 18

19 20 X_(Rol) 21 22 23 2_(pha)

X_(Rol/pha) X_(Rol) 24 3_(pha) X_(Rol/pha) X_(Rol) 25 26 27 28 29 30

SHOT EXPERIMENT: 7

[0077]C. elegans-strain: pha-1 (e2123)

[0078] Marker-DNA:

[0079] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0080] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0081] Test-DNA: pH1-FM6.9 (sud-1) after crossing with vab-9 sud-1

[0082] Addition of nucleic acid:—Status: concluded TABLE 4 results ofshot experiment 7 current 1. selection (rol-6) Test-DNA (sud-1) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1 X_(Rol) 2 3 X_(Rol)4 X_(Rol) 5

X 6 7 3_(pha)

X_(Rol/pha) X 8

4_(pha) X_(Rol/pha) X 9 2_(pha) 3_(pha) X_(Rol/pha) X_(Rol/pha) X X 10

X 11 2_(pha) X_(Rol/pha) X_(Rol) 12

13 X_(Rol) 14 15 3_(pha) X_(Rol/pha) X_(Rol) X 16 X_(Rol) 17 2_(pha)X_(Rol/pha) X 18 X_(Rol) 19 20

X_(Rol)

SHOT EXPERIMENT: 8

[0083]C. elegans-strain: pha-1 (e2123)

[0084] Marker-DNA:

[0085] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0086] 2. Selection with pBX (pha-1) ; together F2/3 at 25° C.

[0087] Test-DNA: pH1-FM6.9 (sud-1) after crossing with vab-9 sud-1

[0088] Addition of nucleic acid:—Status: concluded TABLE 5 results ofshot experiment 8 cur- rent 2. selection shot 1. selection (rol-6)Test-DNA (sud-1) (pha-I) no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3

4

_(pha) 2_(pha) X_(Rol/) pha 5

_(pha) 5_(pha) X_(Rol/) pha 6

_(pha) 7 8 9 10 11

_(pha) 12 13 14 15 16 17 18

_(pha) 19 20

SHOT EXPERIMENT: 9

[0089]C. elegans-strain: pha-1 (e2123)

[0090] Marker-DNA:

[0091] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0092] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0093] Test-DNA: pFM (ceh-13::lacZ) fluorescence in F2/3

[0094] Addition of nucleic acid:—Status: concluded TABLE 6 results ofshot experiment 9 current 1. selection (rol-6) Test-DNA (ceh-13) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3

_(pha) 4

_(pha)

_(pha) 5 6

7 8 3_(pha) X_(Rol/pha) 9

_(pha) 10 11 12 13 14 15 16

17

_(pha)

18 19

_(pha)

20

SHOT EXPERIMENT: 10

[0095]C. elegans-strain: pha-1 (e2123)

[0096] Marker-DNA:

[0097] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0098] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0099] Test-DNA: pFM (ceh-13::lacZ) fluorescence in F2/3

[0100] Addition of nucleic acid: tRNA Status: concluded TABLE 7 resultsof shot experiment 10 current 1. selection (rol-6) Test-DNA (ceh-13) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3_(pha)X_(Rol/pha) 3

_(pha) 4

_(pha) 5 6 7

_(pha) 8 9

_(pha) 10

_(pha) 11 12 13 14 15

_(pha) 16

_(pha)

_(pha) 17

_(pha) 18 19 2_(pha) X_(Rol/pha) 20

_(pha)

SHOT EXPERIMENT: 11

[0101]C. elegans-strain: pha-1 (e2123)

[0102] Marker-DNA:

[0103] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0104] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0105] Test-DNA: pFM (ceh-13::lacZ) fluorescence in F2/3

[0106] Addition of nucleic acid:—Status: interrupted! TABLE 8 results ofshot experiment 11 current 1. selection (rol-6) Test-DNA (ceh-13) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1 X 2

3 4

X 5

X 6

7 8

X 9

10 11

12 13 14

15

16

X 17

X 18

X 19 X 20

SHOT EXPERIMENT: 12

[0107]C. elegans-strain: pha-1 (e2123)

[0108] Marker-DNA:

[0109] 1. Selection with pRF4 (rol-6); individual F1 at 15° C.

[0110] 2. Selection with pBX (pha-1); together F2/3 at 25° C.

[0111] Test-DNA: pFM (ceh-13::lacZ) fluorescence in F2/3

[0112] Addition of nucleic acid:—Status: interrupted! TABLE 9 results ofshot experiment 12 current 1. selection (rol-6) Test-DNA (ceh-13) 2.selection (pha-1) shot no. 1 2 3 4 1 2 3 4 1 2 3 4 1

2

3 4 5 6 7

8 9

10 11

12

13

14

15 16

17

18

19 20

[0113] Explanation Key to Tables: cur- rent shot 1. selection (rol-6)Test-DNA (xxx-xx) 2. selection (pha-1) no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2

3 3

X_(Rol/) pha 4 5 X 6

_(pha) 7 8

4_(pha) X X_(Rol) 9 10

[0114] First (Rol-6) and second (pha-1) selections were carried out onthe same secondary plates 1-4, however, both selection procedures areindependent of one another.

selection path not evaluated or carried out Shot No. 2 On one of foursecondary plates 2 F1 animals were identified with phenotype Rol-6 andisolated; they produced no Rol-6 offspring (F2- F4); transienttransformation Shot No. 3 On two of four secondary plates 3 and 2 F1animals were identified with phenotype Rol-6 and isolated; they producedRol-6 offspring in case 3/2 and no Rol-6 offspring in case 3/4 (F2-F4);stable and transient transformation Shot No. 6 On one of four secondaryplates 2 F1 animals were identified with phenotype Rol-6 and isolated;they produced no Rol-6 offspring (F2-F4) but exhibited phenotypicalrescue of pha-1; transient transformation re Rol-6 but stabletransformation re pha-1 Shot No. 5 On one of four secondary plates,under selection conditions (= 25° C.), phenotypical rescue of pha-1 wasobserved in F2 and F3 animals; stable after several generations; stabletransformation Shot No. 8 On two of four secondary plates 4 and 3 F1animals were identified with phenotype Rol-6 and isolated; in case 8/2they produced no Rol-6 offspring and in case 8/3 they produced Rol-6offspring (F2-F4); moreover, co-transformation was also carried out repha-1; stable and transient transformation On two of four secondaryplates, under selection conditions (= 25 ° C.), phenotypical rescue ofpha-1 was observed in F2 and F3 animals; stable after severalgenerations; in one case (8/3) stable co- transformation re Rol-6 wasevident; stable co-transformation re pha-1 and Rol-6 In case 8/2transiently transformed Rol-6 offspring refer to co- transformation repha-1; In case 8/3 stable transformation re Rol-6 could be verified overboth selection paths. Shot No. 3 Stably transformed lines wereinvestigated further regarding the co-transformation re a particularDNA-test species; in ceh-13:: gfp (pFM) by epifluorescence and in sud-1(pH1-FM6.9) by crossing with vab-9 sud-1 (t-1237); in each case 5 Rol-6positive animals were (pFM) by epifluorescence and in sud-1 (pHl-FM6.9)by crossing with vab-9 sud-1 (t-1237); in each case 5 Rol-6 positiveanimals were tested in this respect; positive re DNA test

[0115] TABLE 10 A rol-6 selection F1 cotransformation # of shots indep.stable ceh-13:: Series shots negative transformants transform.transform. pha-1 GFP sud-1 (9) #1 20 12 24 12 1 1 1 — (10) #2 20 9 25 122 2 2 — (8) #3 20 14 23 8 2 2 — 2 (7) #4 20 10 44 17 7 7 — 7 B rol-6selection F1 pha-1 selection F2 # of shots indep. stable cotransform.shots stable cotransform. Series shots negative transformants transform.transform. pha-1 negative transform. rol-6 (7) #4 20 10 44 16 7 7 4 17 9(6) #5 30 12 56 23 3 3 22 8 5 (1) #6 18 4 52 21 3 3 6 15 13

[0116] Table 10 shows the results of transformation of C. elegans byparticle bombardment.

[0117] Pha-1 (e2123) hermaphrodites were used in all experiments. (A)animals were transformed with the plasmids pRF4 (rol-6) and pBX (pha-1).In addition, a third DNA was tested for its co-expression. A ceh-13::GFPconstruct was properly expressed in transgenic embryos as revealed byfluorescence microscopy (see Wittman et al., as described above). Amaternal effect mutation of sud-1 (t1237) was also complemented by theco-transformed plasmid pH1-FM6.9 (described above) in the transgenes asrevealed by test crosses. Currently about half the shots do not revealany transformants. Transformation events are clustered (Table Y)probably due to single transformed hermaphrodites. (transformants)indicates the number of all Rol animals; (indep transformants) indicatesRol animals derived from different slices of the original plate shot atand thus animals which arose independently. See also (B) for more dataabout the rol-6 selection. (B) in this series of experimentstransformants were also selected in the F2 with the pha-1 system.Cotransformed stable lines were isolated in about half of the shots.Because of the clustering observed with rol-6 it is not clear if theselines represent single events. Therefore lines should be clones out toestablish isogenic lines. TABLE 11 Shot rol-6 selection pha-1 selectionNo. 1 2 3 4 1 2 3 4 1 1R 2 3 1R P 4 5 4R PR P PR PR 6 2R 7 2R 2R PR PR 8 2RP PR 9 PR 10 2R 11 2R 12 1R PR 13 1R 1R 14 12R  15 16  7RP PR 17 1R10RP PR PR PR 18 1R

[0118] Table 11 shows analysis of the shots of series #6. Allexperiments were evaluated as shown here. The worms corresponding to ashot are distributed to 4 plates for selection of transgenic animals asdescribed in part (C) of Example 2. (R) rolling transformant. (P) pha-1transformant. Normal font indicates transient expression of the rolmarker. The numbers of animals found in the Rol selection is indicated.The animals from a slice were reared together to test stable expressionof the rol marker. These lines were then tested for co-transformationwith pha-1. Bold font indicates stable expression of the markers.

EXAMPLE 3 Alternative Technique for Ballistic Transformation

[0119] The worms are prepared for ballistic transformation according topart (A) of Example 2. A drop of a high concentration solution ofnucleic acid (1 mg/ml or more) is placed onto the worm pillow andallowed to dry. The worms are then bombarded with microprojectiles whichhave not been coated with any nucleic acid according to the protocolgiven in part (B) of Example 2 and transformants are selected.

1. A method of introducing a nucleic acid into a nematode worm that is C. elegans, which method comprises: bombarding the worm with a plurality of microprojectiles under conditions to permit the plurality of the microprojectiles to penetrate the worm, wherein the microprojectiles are coated with the nucleic acid and wherein the worm is not dehydrated.
 2. A method as claimed in claim 1, wherein the nematode worm carries a conditional lethal mutant gene and the nucleic acid comprises a plasmid containing a wild-type equivalent of the conditional lethal mutant gene.
 3. A method as claimed in claim 1, wherein the nucleic acid encodes a dominant phenotypic marker.
 4. A method as claimed in claim 3, wherein the dominant phenotypic marker is Rol-6 or an autonomous fluorescent protein.
 5. A method as claimed in claim 1, which further comprises placing the nematode worm onto a thin, dry bacterial lawn prior to bombarding the worm with the plurality of microprojectiles.
 6. A method as claimed in claim 1, which further comprises placing the nematode worm onto a dry polymer plate prior to bombarding the worm with the plurality of microprojectiles.
 7. A method as claimed in claim 6, wherein the polymer is agar.
 8. A method as claimed in claim 6, which further comprises chilling the nematode worm prior to bombarding the worm with the plurality of microprojectiles.
 9. A method as claimed in claim 1, which further comprises chilling the nematode worm prior to bombarding the worm with the plurality of microprojectiles.
 10. A method as claimed in claim 1, in which the nematode worm is immobilized prior to bombardment with the plurality of microprojectiles.
 11. A method as claimed in claim 10, wherein the nematode worm is immobilized by placing the worm onto a dry polymer plate and chilling the plate on ice prior to bombarding.
 12. A method as claimed in claim 1, wherein the microprojectiles are gold particles or activated glass particles.
 13. A method as claimed in claim 1, which further comprises selecting a transformant worm.
 14. A method as claimed in claim 13, wherein selecting a transformant worm comprises selecting a stable transformant worm.
 15. A method as claimed in claim 13, which further comprises allowing the transformant worm to produce larvae.
 16. A method as claimed in claim 15, which further comprises allowing the larvae to develop into a progeny nematode worm.
 17. A method for selecting a nematode worm that is C. elegans and that heritably transmits a nucleic acid to a subsequent generation, which method comprises: (a) bombarding said worm with a plurality of microprojectiles under conditions to permit the nucleic acid to be retained in the worm, wherein the microprojectiles are coated with the nucleic acid; (b) selecting a transformant worm; (c) allowing the transformant worm to produce larvae that develop into a progeny worm; and (d) determining whether the progeny worm comprises the nucleic acid. 